Understanding the earliest molecular changes that drive the genesis of type 2 diabetes may lead to the development of new diagnostics, prognostics, and potentially new targets for therapeutic intervention. We have discovered a new member of the basic-helix-loop-helix-zipper family of transcription factors called MondoA that we propose plays a critical role in skeletal muscle glucose homeostasis and insulin resistance. MondoA functions primarily as a transcriptional activator, binding to CACGTG genomic targets with its obligate heterodimeric partner Mlx. Several lines of evidence indicate that MondoA:Mlx complexes are key regulators of cellular bioenergetics. First, MondoA:Mlx complexes are not constitutively nuclear proteins, rather they shuttle between the outer mitochondrial membrane and the nucleus, suggesting that they facilitate communication between these two essential organelles. Second, MondoA:Mlx complexes accumulate in the nucleus, binding their target promoters to activate their expression under hyperglycemic conditions by sensing glucose-6-phosphate levels. Third, MondoA:Mlx complexes are required for transcriptional activation of at least 75 percent of glucose-induced targets, indicating that they are major regulators of the glucose-dependent transcriptome. Finally, MondoA is very highly expressed in skeletal muscle, which is a major site of glucose disposal and is transcriptionally active in a number of muscle cell lines. Together, we suggest that skeletal muscle is one of MondoA's primary sites of action. We hypothesize a critical function for MondoA in how skeletal muscle senses and responds to changes in glucose levels. One critical MondoA target is thioredoxin interacting protein (TXNIP). TXNIP has pleiotropic roles in glucose homeostasis and insulin resistance, which are mediated in part by its function as a negative regulator of peripheral glucose disposal. Consistent with TXNIP being a critical MondoA effector, MondoA is also a potent negative regulator of glucose uptake. As such, we hypothesize that the nuclear activity MondoA and its transcriptional targets may also drive skeletal muscle insulin resistance by negatively regulating glucose uptake. TXNIP is not the sole MondoA effector in attenuating glucose uptake, indicating that other MondoA transcriptional targets must also contribute. We propose to employ a conditional deletion allele of murine MondoA to specifically inactivate MondoA in skeletal muscle. In Aims 1, we will use this mouse model to determine MondoA's in vivo function in skeletal muscle glucose homeostasis and whether MondoA activity is required for skeletal muscle insulin resistance as our preliminary data suggest. Further, we propose comprehensive genomic approaches in Aim 2 to discover the direct and MondoA-dependent transcriptome in skeletal muscle. Finally, our preliminary data indicate that MondoA transcriptional activity is controlled by mitochondrial overload. We will test this model in Aim 3 and determine how MondoA and TXNIP function as potent negative regulators of glucose uptake. PUBLIC HEALTH RELEVANCE: The transcriptional regulator MondoA is highly expressed in skeletal muscle and is a master regulator of glucose-induced transcription. MondoA is a potent negative regulator of glucose uptake in a number of different cell lineages via its positive transcriptional regulation of thioredoxin interacting protein (TXNIP). However, TXNIP is not the sole MondoA effector in this regard. In this application, we propose a mouse model to determine the function of MondoA in skeletal muscle and determine whether MondoA is necessary for the development of insulin resistance or diabetes. Furthermore, we propose comprehensive genomics approaches to determine the direct and glucose-induced MondoA transcriptome in skeletal muscle, which will provide insight into the transcriptional programs that drive insulin resistance. Finally, we propose approaches to how mitochondrial dysfunction controls MondoA transcriptional activity